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While phylogenetic studies have shown how WNV has diversified and undergone genotype replacement since introduction, more geographically focused studies are needed to understand intricate transmission dynamics at local and regional scales. In this study, we validate the IDT xGen WNV panel, a novel single reaction amplicon-based Next-Generation Sequencing approach, to generate high-quality WNV genomes and compare it to the \u201cPrimal Scheme\u201d assay for WNV, a common amplicon sequencing strategy. By generating &gt;250 genomes from mosquito pools, we show that the IDT xGen WNV panel generated coding-complete and accurate WNV genomes when compared to the current sequencing approaches. Additionally, we used this approach to generate 100 coding-complete WNV genomes from surveillance pools of mosquitoes collected in Nebraska during the 2023 outbreak. Our discrete phylogeographic analysis revealed substantial genetic diversity in WNV genomes from 2023 with minimal clustering across the state. This study demonstrated the utility of a single reaction amplicon-based sequencing approach to generate quality WNV genomes from routine surveillance samples and characterize WNV transmission dynamics in a high-incidence setting.<\/jats:p>","DOI":"10.1371\/journal.pntd.0013931","type":"journal-article","created":{"date-parts":[[2026,1,21]],"date-time":"2026-01-21T18:42:22Z","timestamp":1769020942000},"page":"e0013931","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Genomic characterization of a severe West Nile Virus transmission season using a single reaction amplicon sequencing approach"],"prefix":"10.1371","volume":"20","author":[{"given":"Shawn","family":"Freed","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah","family":"Chandler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah","family":"Uhm","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zach","family":"Pella","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dikchha","family":"Gurung","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hallie","family":"Smith","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tammy","family":"Dowdy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amanda M.","family":"Bartling","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ava","family":"Butz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael R.","family":"Wiley","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M. 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